The present invention is related to optical detection systems. In particular, the present invention is related to optical detection systems which can analyze multiple samples simultaneously.
On many occasions, in chemistry and biology, large numbers of samples need to be analyzed. Particularly in molecular biology, in the Human Genome Project, high speed analyses with high throughput are necessary to achieve the goals of the project. Genetic mapping and DNA sequencing on slab gels are currently performed by using automated DNA sequencing with mono-colour or multi-color fluorescent dye labeling. Because capillary electrophoresis (CE) and particularly CE combined with laser induced fluorescence (CE-LIF) offers rapid charged species analyte separation and high detection sensitivity, it is particularly attractive as a separation technique in DNA sequencing applications. However, the number of capillaries that can be analyzed at one time limits the total throughput of the analysis. To increase the throughput a technique called capillary array electrophoresis (CAE) has been introduced. In this technique multiple capillaries are used in parallel with some advantages over slab gels with multiple lanes. There is a substantial reduction on Joule heating effect. Therefore, higher electric fields can be applied and faster analysis can be obtained. The cost of material is reduced in terms of gel usage due to the reduced diameter of the capillaries, as well as samples usage due to a smaller sample size. Another advantage is the possibility to increase the sample throughput by increasing the number of channels in theory up to thousands, while the slab gels impose physical size and sample loading difficulties.
Various methods for acquiring signals from multiple channels have been described; however, the simultaneous detection of the different channels in CAE still presents some problems. A multiple capillary electrophoresis laser induced fluorescence detector that utilizes a confocal fluorescence scanner is described in U.S. Pat. Nos. 5,091,652 and 5,274,240. The scanner or computer controlled stage translates the capillary array past the light path of a laser beam and the optical detection system. Since relatively heavy components are being moved problems with misalignments of the capillaries relative to the light source are likely to occur. To avoid problems derived from the movement of bulky components in U.S. Pat. No. 5,675,155, a detection system is described where an excitation laser beam is focused and scanned across the is capillary array by the movement of a mirror which is aligned as well to receive the electromagnetic radiation from the sample. The advantages of these approaches are the use of a small local illumination and detection volumes requiring only modest excitation power for optimal signal to noise ratio. Crosstalk between adjacent capillaries is eliminated since only a single capillary is illuminated at a time. On the other hand, because the data acquisition is sequential, i.e., the scan modes are from the first capillary to the last capillary in the array, the use for a very large number of capillaries is limited by the observation time needed per capillary. Loss of information could happen in case of a large number of capillaries.
Another multiplexed detector system for capillary electrophoresis is described in U.S. Pat. No. 5,498,324. The invention involves laser irradiation of the sample in a plurality of capillaries through individual optic fibers inserted into the outflow of each capillary. Quesada and Zhang (Electrophoresis 17, 1841-1851, 1996) improved this design by using fiber optics for illumination and collection of the fluorescent emission orthogonally. One of the advantages of the this approach is that no moving parts are involved. However, in both systems the excitation energy that reaches each capillary does not have a homogeneous distribution and degrades as the numbers of fibers included in the fused taper splitter increases. In addition, detection of the arrays is simultaneous through a CCD combined with microscope or camera lens. Therefore, in this case the limitation of the number of capillaries that can be detected at one time depends on the number of them that can be packed in the imaging field of the detector and the resolution of the detector. The most critical problem in this approach may be cross talk between capillaries because fluorescence from adjacent capillaries can be refracted to reach the detector. Although cross talk between capillaries can be avoided by the use of spacers, it is evident that the use of spacers will reduce the number of capillaries in the array.
A greater number of capillaries can be measured at the same time (U.S. Pat. No. 5,730,850) by arranging capillaries two-dimensionally in a capillary array sheet and using a simultaneous two-dimensional detector. Employing modified sheath-flow cuvette detection, sensitivity is enhanced by eliminating light interferences. However, the simultaneous illumination of all the capillaries requires a complicated system of mirrors to transmit the light beam through the buffer solution path between the capillary holder and the detection window, which in turn may result in differences in intensity.
Accordingly, it is desirable to provide an economical and high sensitive detection system for multiple sample analysis which is easy to set up and easy to handle where bulky moving parts and complicated alignments are minimized.
It is therefore an object of the present invention to provide a system to overcome the shortcomings as stated above.
It is another object to provide an optical detection system which allows only collimated light to reach the sample to be detected.
It is a further object to provide an embodiment of an optical detection system which can perform simultaneous detection in a plurality of samples with reduced scattering and cross-talk.
The present invention is an optical detection system comprising an electromagnetic radiation source, a source radiation focusing and collimating means, a photodetector, an emitted radiation focusing means and a source radiation blocking panel. The radiation source is used to direct source radiation onto a sample which is disposed in a sample platform. The source radiation focusing and collimating means is disposed between the radiation source and the sample for focusing and collimating the source radiation onto the sample. The photodetector is adapted for receiving radiation emitted from the sample which has been focused by the emitted radiation focusing means. The source radiation blocking panel, disposed between the source radiation focusing and collimating means and the sample, is unique in that it is capable of reducing light scattering and interference, such that a clear signal from each individual sample can be obtained by the photodetector.
In the most preferred embodiment, the source radiation focusing and collimating means comprises at least one convergent cylindrical rectangular lens and the source radiation blocking panel comprises a light absorbing panel with at least one pinhole. The samples are contained in channels or tubes aligned in parallel. For simplicity, the samples contained in the various channels or tubes are referred to as sample volumes. In one embodiment, the emitted radiation focusing means is a convex lens, while in another embodiment, it is a convergent cylindrical lens together with an emitted radiation blocking panel having pinholes. This panel with pinholes will be referred to simply as pinholes in the following description. The pinholes may be connected to scanning or conveying means to allow movement. The system may be used for the detection of radiation absorbance or for fluorescence, including epi-fluorescence. Static pinholes for reducing interference and moving pinholes for sequentially and repetitively illuminating selected sample volumes from an array of samples. In the cases of no cross talk between samples or when cross talk can be eliminated, static pinholes are used to reduce interference due to scattered light, while moving pinholes can be used to eliminate cross talk between samples by sequentially and selectively illuminating only the sample volumes to be measured at any instant of time. In this embodiment, the system includes a plurality of sample volumes in parallel comprising: an array of channels, capillaries, flow cells, bands or wells; at least one electromagnetic radiation source; at least one convergent rectangular cylindrical lens to focus electromagnetic radiation; at least one set of static pinholes or moving pinholes; a scanner for moving the pinholes; and at least one detector aligned to receive electromagnetic radiation collected from the sample volumes. The pinholes are placed in between the array of samples and the detector and for between the array of samples and the electromagnetic radiation source. For operation of the system using static pinholes, the number of pinholes should match that of the samples in the array. The electromagnetic radiation energy that reaches each sample volume is homogeneously focused and distributed by the convergent rectangular cylindrical lens through the array of pinholes. Emitted electromagnetic radiation from all of the sample volumes is collected and directed to a detector simultaneously. Pinholes are used to prevent scattered electromagnetic radiation from reaching the detector. In operation of the system using moving pinholes, the number of pinholes is less than that of the samples in the array and can be as few as one. Only the electromagnetic radiation energy that can pass through the pinholes can reach selected sample volumes. The scanner for moving the pinholes adjusts the position of the pinholes so that only selected sample volumes are illuminated by the electromagnetic radiation. Emitted electromagnetic radiation from the selected sample volumes is collected and directed to a detector where a signal is generated in response to the interaction of the electromagnetic radiation with the sample. This operation is performed sequentially and repetitively with each sample volume in the array. Moving pinholes are also used to prevent scattered electromagnetic radiation from reaching the detector. Advantageously, the present invention provides two detection systems for multiple sample analysis, which are easy to set up and easy to handle where bulky moving parts and complicated alignments are minimized, and allows the electromagnetic radiation source to remain on selected sample volumes for a preset period of time. The result is higher sample throughput, improved detection sensitivity and more economical and physically stable detection systems.
In preferred embodiments, the sample is imaged from an array of channels microfabricated in glass, quartz, fused silica or polymeric materials for capillary electrophoresis. In one embodiment, the source radiation is an excitation light, and the the sample in each channel is fluorescent or contains a fluorescent label and is separated on an electrophoretic medium, or the sample is not fluorescent and is separated in a fluorescent electrophoretic medium. The electromagnetic radiation source preferred is a laser but other right sources, mercury lamps, xenon lamps or any other light sources with the appropriated power and wavelength can be used. The source radiation wavelength specific to the sample to be investigated is isolated by interference filter and transmitted axially to the sample. The source radiation is focused linearly by a convergent rectangular cylindrical lens. The focal distance between the lens and the channels is adjusted manually by movement of a translational stage in the x, y and z directions or by an auto-focusing system. In the same direction the fluorescent emission is collected and collimated by the lens through the array of pinholes or by moving pinholes. A long pass filter is selected to block wavelengths below the emission. An array of pinholes or moving pinholes can be used to prevent non-collimated light from reaching the detector.
The present invention provides detection systems with which a plurality of sample volumes can be analyzed. In consequence, this system allows for a significant increase in throughput of batches of samples. Different types of optical detection systems can be used, such as visible, ultraviolet or fluorescent in the preferred embodiments, we will refer to CE-LIF (laser induced fluorescence), because of its higher sensitivity, performed in microfabricated channels. For those skilled in the art, it is well known that the system is equally applicable for capillary electrophoresis in fused silica capillaries, and since this system is provided with a focusing facility, any coplanar, linear and closely distributed samples can be easily incorporated into the field of view and optically analyzed by the detector. In addition, this multichannel detection system can be used in the analysis of chemicals, such as ions and drugs, or bio-molecules, such as DNA, RNA, proteins, viruses, bacteria and the like by HPLC or other analytical techniques involving the use of capillaries, microchannels, flow cells, bands or wells. In general it can be useful for optical testing of series of homologous samples volumes distributed closely in reservoirs and in the same plane.